Plant Molecular Biology

, Volume 83, Issue 4–5, pp 391–404 | Cite as

Creation and validation of a widely applicable multiple gene transfer vector system for stable transformation in plant

Article

Abstract

Multiple gene transfer (MGT) technology has become a powerful tool for basic and applied plant biology research in recent years. Despite some notable successes in obtaining plant lines harbouring multiple transgenes, these methods are still generally unwieldy and costly. We report here a straightforward and cost effective strategy, utilizing commonly available restriction enzymes for the transfer of multiple genes into plants, hence greatly widening the accessibility of MGT. This methodology exploits the specific ‘nested’ arrangement of a pair of isocaudomer restriction enzymes (for example XbaI—AvrII–XbaI) so that through the alternate use of these two enzymes in a reiterative fashion multiple genes/constructs (up to five in this study) could be ‘stacked’ together with ease. In a proof-of-concept experiment, we constructed a plant transformation vector containing three reporter gene expression cassettes flanked by two matrix attachment region sequences. The expression of all three genes was confirmed in transgenic Arabidopsis thaliana. The usefulness of this technology was further validated by the construction of a plant transformation vector containing five transgenes for the production of eicosapentaenoic acid (EPA, C20∆5,8,11,14,17), a polyunsaturated essential fatty acid found in fish oils that is beneficial for health. In addition, we constructed four more vectors, incorporating one seed specific and three promoters conferring constitutive expression. These expression cassettes are flanked by a different isocaudomer pair (AvrII—SpeI–AvrII) and four other unique restriction sites, allowing the exchange of promoters and terminators of choice.

Keywords

Multiple gene transfer Isocaudomer Transgenic plant EPA 

Abbreviations

AA

Arachidonic acid

ALA

α-Linolenic acid

DHA

Docosahexaenoic acid

D8

Δ8 Desaturase gene

D5

Δ5 Desaturase gene

D15

Δ15 Desaturase gene

D17

Δ17 Desaturase gene

E9

Δ9 Elongase gene

EDA

Eicosadienoic acid

EPA

Eicosapentaenoic acid

ETA

Eicosatetraenoic acid

ETrA

Eicosatrienoic acid

GFP

Green fluorescent protein

GUS

β-Glucuronidase gene

LA

Linoleic acid

MARs

Matrix attachment regions

MCS

Multiple cloning site

MGT

Multiple gene transfer

MISSA

Multiple-round in vivo site-specific assembly

MS

Murashige and Skoog

PEC

Plant expression cassette

tNOS

Nos terminator

VLCPUFAs

Very long chain polyunsaturated fatty acids

ZFNs

Zinc finger nucleases

35S

CaMV 35S promoter

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.State Key Laboratory of Crop BiologyShandong Agricultural UniversityTaianChina
  2. 2.Shandong Peanut Research InstituteQingdaoChina

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